Clearing agent and mounting media for microscopy

ABSTRACT

A clearing agent and mounting solution for microscopy is disclosed comprising at least trichloroethanol and/or derivatives thereof, where the refractive index of the solution is greater than or equal to about 1.3810. Also disclosed are methods of preparing specimens for microscopy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation-in-Part of U.S. application Ser. No.15/258,474, filed on Sep. 7, 2016, and also a Continuation-in-Part ofU.S. application Ser. No. 14/391,106, filed on Oct. 7, 2014; U.S.application Ser. No. 15/258,474 is a continuation of U.S. applicationSer. No. 14/391,106, filed on Oct. 7, 2014, which is a 371 NationalPhase of PCT/US2013/035761 filed Apr. 9, 2013, which claims the benefitof priority to U.S. Application No. 61/622,210 filed Apr. 10, 2012, theentire disclosures of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present application is related to microscopy, and more particularly,to a clearing agent and mounting media for microscopy.

BACKGROUND

There are different methods for identification of materials, includingmacroscopic, chemical and microscopic identification, among others.Microscopic identification is a technique that uses a microscope toidentify characteristic features of living organisms, parts of anorganism, cells or sub-cellular organs, as well as minerals or othernon-living materials. The importance of microscopy resides in theability to clearly identify differences between organisms or their partsby focusing on specific characteristics or diagnostic structures.Microscopy relies on dependable, readily available reagents as clearingagent and mounting solutions, optionally used in conjunction with stainsin order to visualize the materials under the slide.

The general microscopy procedure for specimens derived from livingorganisms comprises mounting a small sample of the tissue to be analyzedin a solvent solution and observing it under the microscope. In manycases the cell contents obscure the tissues, making it difficult toidentify characteristic features. Differences in refractive index withinthe specimen prohibit visualization of deeper visual planes, and occludedetail from observation. In these cases a clearing solution is appliedin order to improve the transparency of the specimen, allowing one tovisualize multiple vertical layers of the specimen without carefulsectioning or remounting. This increased transparency and improvedclarity allows the microscope user to visualize across a full range ofvertical planes in the sample, allowing the user to select interestingfocal planes by adjusting the focus.

An almost universally used clearing agent for microscopy is acidifiedchloral hydrate glycerol solution (chloral hydrate solution acidifiedwith hydrochloric acid), also known as Hertwig's solution. Acidifiedchloral hydrate solution is used in botanical microscopy, mycology,entomology, histology, mineralogy, food science, quality control,forensics, nematology, archeology, paleontology, virology, immunology,microscopy including, but not limited to, differential interferencecontrast microscopy, electron microscopy, fluorescence microscopy,confocal microscopy, and other related applications of microscopy andoptics. Chloral hydrate, when applied to botanical samples, dissolvescellular contents and intercellular substances thus allowing cell wallsand shapes of the cells to be easily observed. Consequently, chloralhydrate has become the industry standard for many laboratories focusedon quality assessment of herbal products.

Unfortunately, chloral hydrate, the key component in acidified chloralhydrate solution, is considered under US law to be a narcotic hypnotic,and as such is a DEA (Drug Enforcement Administration) scheduledsubstance, requiring DEA approval and compliance in order to purchaseand/or possess it. This has precluded scientists from being able topurchase this reagent. Furthermore, maintaining DEA compliance is acostly, tedious, and time-consuming process.

Therefore, cost-effective, readily available, and unregulatedreplacements for acidified chloral hydrate solution are needed asclearing and mounting agents for microscopy.

SUMMARY

It has now been discovered that aqueous solutions of trichloroethanol,or its derivatives or analogs, with or without other additives, caneffectively replace acidified chloral hydrate solution as a clearing andmounting medium for microscopy.

This discovery has been exploited to develop the present disclosure,which, in one aspect, is the clearing agent and mounting solutioncomprising 10% to 97% (v/v) trichloroethanol or derivative thereof. Insome embodiments, the mounting solution and clearing agent furthercomprises 16.5% to 36% (v/v) benzyl alcohol, 12.5% to 50% glycerol,polyethylene glycol (PEG), propylene glycol (PG), ethylene glycol (EG),and/or derivatives thereof, and optionally water.

In yet another aspect, a clearing agent and mounting solution comprising5% to 97% (v/v) trichloroethanol or derivative thereof 16.5% to 36%(v/v) benzyl alcohol; 12.5% to 50% glycerol, polyethylene glycol (PEG),propylene glycol (PG), ethylene glycol (EG), and/or derivatives thereofand optionally water.

In some embodiments, the clearing agent and mounting solution comprises36% (v/v) of trichloroethanol or derivative thereof; 36% (v/v) benzylalcohol; and 27% (v/v) glycerol. In other embodiments, the clearingagent and mounting solution comprises 33% (v/v) of trichloroethanol orderivative thereof; 33% (v/v) benzyl alcohol; and 33% (v/v) glycerol. Inyet other embodiments, the clearing agent and mounting solutioncomprises 33% (v/v) trichloroethanol or derivative thereof; 33% (v/v)benzyl alcohol; 17% (v/v) glycerol; and 17% (v/v) benzyl benzoate. Instill other embodiments, the clearing agent and mounting solutioncomprises 29% (v/v) trichloroethanol or derivative thereof 29% (v/v)benzyl alcohol; 17% (v/v) glycerol; 13% (v/v) benzyl benzoate; and 13%(v/v) benzyl ether.

In certain embodiments, a clearing agent and mounting solution comprises42% (v/v) trichloroethanol or derivative thereof; 16.5% (v/v) benzylalcohol; 16.5% (v/v) glycerol; and 25% (v/v) benzyl ether. In otherembodiments, the clearing agent and mounting solution comprises 36%(v/v) of trichloroethanol or derivative thereof; 36% (v/v) benzylalcohol; and 27% (v/v) PEG, PG, EG, and/or derivatives thereof. In yetanother embodiment, the clearing agent and mounting solution comprises33% (v/v) of trichloroethanol or derivative thereof; 33% (v/v) benzylalcohol; and 33% (v/v) PEG, PG, EG, and/or derivatives thereof. In stillother embodiments, the clearing agent and mounting solution comprises33% (v/v) of trichloroethanol or derivative thereof; 33% (v/v) benzylalcohol; 17% (v/v) PEG, PG, EG. and/or derivatives thereof; and 17%(v/v) benzyl benzoate.

In some embodiments, the clearing agent and mounting solution comprise29% (v/v) of trichloroethanol or derivative thereof; 29% (v/v) benzylalcohol; 17% (v/v) PEG, PG, EG. and/or derivatives thereof 13% (v/v)benzyl benzoate; and 13% (v/v) benzyl ether. In certain embodiments, theclearing agent and mounting solution comprises 42% (v/v)trichloroethanol or derivative thereof; 16.5% (v/v) benzyl alcohol;16.5% (v/v) PEG, PG, EG. and/or derivatives thereof and 25% (v/v) benzylether. In other embodiments, the trichloroethanol derivative comprisesmono- or poly-halogenated branched or unbranched alcohols, diols,glycols, aldehydes, aldehyde-hydrates, hemi-acetals, acetals, ketals,aminals, and hemi-aminals of at least 2 carbon units, where branches aredefined as any mono- or poly-halogenated aliphatic or aromatic sidechains containing hydroxyl, amino, ether, carboxyl, carboxyamido,carbonate, carbamyl, carbonyl-chloride, polyethylene glycol, oraminoethanol groups, and a polymer thereof. In other embodiments, theclearing agent and mounting solution further comprises C1-C6 alcohol. Inyet other embodiments, the clearing agent and mounting solution furthercomprises a solidification aid comprising polyethylene glycol, polyamideresin, polyvinylpyrrolidone, polyvinyl alcohol, or mixtures thereof.

In another aspect, the disclosure provides a clearing agent and mountingsolution comprising 5% to 90% (v/v) trichloroethanol or derivativethereof; and 5% to 90% (v/v) benzyl alcohol. In some embodiments, theclearing agent and mounting solution comprises 10% to 90% (v/v)trichloroethanol or derivative thereof and 10% to 90% (v/v) benzylalcohol. In other embodiments, the clearing agent and mounting solutionfurther comprises PEG. EG, PG, and/or derivatives thereof. In yet otherembodiments, the clearing agent and mounting solution further comprisesC1-C6 alcohol. In still other embodiments, the clearing agent andmounting solution further comprises a solidification aid comprisingpolyethylene glycol, polyamide resin, polyvinylpyrrolidone,polyvinylalcohol, or mixtures thereof. In certain embodiments, thetrichloroethanol derivative comprises mono- or poly-halogenated branchedor unbranched alcohols, diols, glycols, aldehydes, aldehyde-hydrates,hemi-acetals, acetals, ketals, aminals, and hemi-aminals of at least 2carbon units, wherein branches are a mono- or poly-halogenated aliphaticor aromatic side chains comprising hydroxyl, amino, ether, carboxyl,carboxyamido, carbonate, carbamyl, carbonyl-chloride, polyethyleneglycol, or aminoethanol groups, and polymers thereof.

In yet another aspect, the disclosure provides a clearing agent andmounting solution comprising 5% to 90% (v/v) trichloroethanol orderivative thereof; and 5% to 90% (v/v) benzyl benzoate. In someembodiments, the clearing agent and mounting solution comprises 10% to90% (v/v) trichloroethanol or derivative thereof; and 10% to 90% (v/v)benzyl benzoate. In other embodiments, the clearing agent and mountingsolution further comprises PEG. EG, PG, and/or derivatives thereof. Inyet other embodiments, the clearing agent and mounting solution furthercomprises C1-C6 alcohol. In still other embodiments, the clearing agentand mounting solution further comprises a solidification aid comprisingpolyethylene glycol, polyamide resin, polyvinylpyrrolidone,polyvinylalcohol, or mixtures thereof. In certain embodiments, thetrichloroethanol derivative comprises mono- or poly-halogenated branchedor unbranched alcohols, diols, glycols, aldehydes, aldehyde-hydrates,hemi-acetals, acetals, ketals, aminals, and hemi-aminals of at least 2carbon units, wherein branches are a mono- or poly-halogenated aliphaticor aromatic side chains comprising hydroxyl, amino, ether, carboxyl,carboxyamido, carbonate, carbamyl, carbonyl-chloride, polyethyleneglycol, or aminoethanol groups, and polymers thereof.

In still another aspect, the disclosure provides a clearing agent andmounting solution comprising 5% to 90% (v/v) trichloroethanol orderivative thereof; 5% to 90% (v/v) benzyl alcohol; and 5% to 90% (v/v)benzyl benzoate. In some embodiments, the clearing agent and mountingsolution comprises 10% to 90% (v/v) trichloroethanol or derivativethereof; 10% to 90% (v/v) benzyl alcohol; and 10% to 90% (v/v) benzylbenzoate. In other embodiments, the clearing agent and mounting solutioncomprises 25% to 50% (v/v) trichloroethanol or derivative thereof; 25%to 50% (v/v) benzyl alcohol; and 33% (v/v) benzyl benzoate. In yet otherembodiments, the clearing agent and mounting solution comprises 33%(v/v) of trichloroethanol or derivative thereof; 33% (v/v) benzylalcohol; and 33% (v/v) benzyl benzoate. In still other embodiments, theclearing agent and mounting solution further comprises PEG. EG, PG,and/or derivatives thereof. In certain embodiments, the clearing agentand mounting solution further comprises C1-C6 alcohol. In someembodiments, the clearing agent and mounting solution further comprisesa solidification aid comprising polyethylene glycol, polyamide resin,polyvinylpyrrolidone, polyvinylalcohol, or mixtures thereof. In otherembodiments, the trichloroethanol derivative comprises mono- orpoly-halogenated branched or unbranched alcohols, diols, glycols,aldehydes, aldehyde-hydrates, hemi-acetals, acetals, ketals, aminals,and hemi-aminals of at least 2 carbon units, wherein branches are amono- or poly-halogenated aliphatic or aromatic side chains comprisinghydroxyl, amino, ether, carboxyl, carboxyamido, carbonate, carbamyl,carbonyl-chloride, polyethylene glycol, or aminoethanol groups, andpolymers thereof.

In another aspect, the disclosure provides a clearing agent and mountingsolution comprising 5% to 90% (v/v) of trichloroethanol or derivativethereof; and 5% to 90% (v/v) benzyl ether. In some embodiments, theclearing agent and mounting solution comprises 25% (v/v) oftrichloroethanol or derivative thereof and 25% (v/v) benzyl ether. Inother embodiments, the clearing agent and mounting solution comprises50% (v/v) of trichloroethanol or derivative thereof; and 50% (v/v)benzyl ether. In yet other embodiments, the clearing agent and mountingsolution further comprises 25% (v/v) benzyl alcohol; and 25% (v/v)benzyl benzoate. In still other embodiments, the clearing agent andmounting solution comprises 33% (v/v) of trichloroethanol or derivativethereof 33% (v/v) benzyl ether; and 33% (v/v) benzyl alcohol. In certainembodiments, the clearing agent and mounting solution further comprisesPEG. EG, PG, and/or derivatives thereof. In other embodiments, theclearing agent and mounting solution further comprises C1-C6 alcohol. Inyet other embodiments, the clearing agent and mounting solution furthercomprises a solidification aid comprising polyethylene glycol, polyamideresin, polyvinylpyrrolidone, polyvinylalcohol, or mixtures thereof. Instill other embodiments, the trichloroethanol derivative comprises mono-or poly-halogenated branched or unbranched alcohols, diols, glycols,aldehydes, aldehyde-hydrates, hemi-acetals, acetals, ketals, aminals,and hemi-aminals of at least 2 carbon units, wherein branches are amono- or poly-halogenated aliphatic or aromatic side chains comprisinghydroxyl, amino, ether, carboxyl, carboxyamido, carbonate, carbamyl,carbonyl-chloride, polyethylene glycol, or aminoethanol groups, andpolymers thereof.

In another aspect, the disclosure provides a method of preparing aspecimen for microscopy, comprising soaking a specimen in sufficientquantity of a clearing solution according to the disclosure for at leastabout 1 minute to about 12 months to provide a cleared specimen;applying the cleared specimen to a microscope slide, cuvette, or wellfor observation; and optionally, applying a cover slip.

In yet another aspect, after soaking the specimen, the cleared specimenis applied to a resin which solidifies, and then is cast into a solidfor examination or indefinite storage.

In another aspect, after soaking the specimen, a solution of 5%-20%polyvinylpyrrolidone in methanol is applied to the cleared specimen,which optionally is dried at 50° C.-90° C. until hardened.

In yet another aspect, after soaking the specimen, a stain or dye isapplied to the cleared specimen to provide a cleared stained specimen,whereby the features of the specimen are selectively highlighted. Thecleared stained specimen is applied to a microscope slide, cuvette, orwell for observation, and optionally, is covered with a cover slip. Thestain or dye can be a fluorescent stain or dye, so that the clearedstained specimen can be visualized using a fluorescent and/orepifluorescent and/or confocal microscope.

Another aspect is directed to a method of preparing a specimen forspectrophotometric analysis, comprising applying a specimen to acuvette; and applying a sufficient quantity of a clearing agent andmounting solution according to the disclosure to mount said specimen.

In another aspect, the disclosure provides a method of preparing aspecimen for microscopy, comprising soaking a specimen in sufficientquantity of a clearing agent and mounting solution according to thedisclosure for at least about 1 minute to about 12 months to provide acleared specimen; applying the cleared specimen to a microscope slide,cuvette, or well for observation; and optionally, applying a cover slip.

Alternatively, in another aspect, after soaking the specimen in aclearing agent according to the disclosure, the cleared specimen isapplied to a resin which solidifies, and then is cast into a solid forexamination or indefinite storage.

In yet another aspect, after soaking the specimen in a clearing agentaccording to the disclosure, a solution of 5%-20% polyvinylpyrrolidonein methanol is applied to the cleared specimen, which optionally isdried at 50° C.-90° C. until hardened.

In still another aspect, after soaking the specimen in a clearing agentand mounting solution according to the disclosure, a stain or dye isapplied to the cleared specimen to provide a cleared, stained specimen,whereby the features of the specimen are selectively highlighted. Thecleared, stained specimen is applied to a microscope slide, cuvette, orwell for observation, and optionally, is covered with a cover slip. Thestain or dye can be a fluorescent stain or dye, so that the clearedstained specimen can be visualized using a fluorescent and/orepifluorescent and/or confocal microscope.

Another aspect of the application is directed to a method of preparing aspecimen for spectrophotometric analysis, comprising applying a specimento a cuvette; and applying a sufficient quantity of a clearing agent andmounting solution according to the disclosure to mount said specimen.

The disclosure also provide a method of preparing a specimen forspectrophotometric analysis wherein, before soaking the specimen in aclearing agent and mounting solution, a stain or dye is applied to thespecimen to provide a stained specimen, whereby the features of thespecimen are selectively highlighted. The stained specimen is thensoaked in the clearing solution according to the disclosure for at leastabout 1 minute to about 12 months to provide a cleared, stainedspecimen. The cleared, stained specimen is applied to a microscopeslide, cuvette, or well for observation, and optionally, is covered witha cover slip. The stain or dye can be a fluorescent stain or dye, sothat the cleared stained specimen can be visualized using a fluorescentand/or epifluorescent and/or confocal microscope.

In still another aspect, the disclosure is directed to use of a clearingagent and mounting solution according to the disclosure to removepigment, dye, stain, or color from a specimen.

A further aspect of the application is directed to use of a clearingagent and mounting solution according to the disclosure to remove excessstain or dye and to increase the contrast of particular structuresand/or organisms within a specimen.

Another aspect of the application is directed to use of a clearing agentand mounting solution to increase the transparency of a specimen and toallow multiple vertical planes to be visualized without the need tosection, remount, or further modify the specimen.

Yet another aspect of the disclosure is directed to use of a clearingagent and mounting solution to simultaneously dehydrate, depigment, andclear specimens for microscopic and/or visual analysis.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects of the present disclosure, the variousfeatures thereof, as well as the invention itself may be more fullyunderstood from the following description, when read together with theaccompanying drawings in which:

FIGS. 1A-1B are representations of microscopic views of a characteristicannular vessel elements and fibers of ginger, Zingiber officinale Roscoe(Zingiberaceae); comparison of acidified chloral hydrate solution versusExample 1 solution;

FIGS. 2A-2B are representations of microscopic views of abundant starchgrains of ginger, Zingiber officinale Roscoe (Zingiberaceae); comparisonof acidified chloral hydrate solution versus Example 1 solution;

FIGS. 3A-3B are representations of microscopic views of ginger epidermisand parenchyma cells, Zingiber officinale Roscoe (Zingiberaceae);comparison of acidified chloral hydrate solution versus Example 1solution;

FIGS. 4A-4B are representations of microscopic views of fragments ofepidermis over leaf veins, Mate (leaves), Ilex paraguariensis(Aquifoliacea); comparison of acidified chloral hydrate solution versusExample 1 solution;

FIGS. 5A-5B are representations of microscopic views of the upperepidermis underlying palisade cells, Mate (leaves), Ilex paraguariensis(Aquifoliacea); comparison of acidified chloral hydrate solution versusExample 1 solution;

FIGS. 6A-6B are representations of microscopic views of fragments orgroups of pericycle of fibers, Mate (leaves), Ilex paraguariensis(Aquifoliacea); comparison of acidified chloral hydrate solution versusExample 1 solution;

FIGS. 7A-7B are representations of microscopic views of the lowerepidermis showing characteristic anomocytic stomata, Mate (leaves), Ilexparaguariensis (Aquifoliacea); comparison of acidified chloral hydratesolution versus Example 1 solution;

FIGS. 8A-8B are representations of microscopic views of rounded orelliptical pollen grains with three germinal pores, exine (outermostcell wall of pollen grain) dentate spinose, Safflower (flower),Carthamus tinctorius L. (Asteraceae); comparison of acidified chloralhydrate solution versus Example 1 solution;

FIGS. 9A-9B are representations of microscopic views of laticiferousducts (tubular cells containing latex fluid) with a reddish-brownsecretion next to vessels elements, Safflower (flower), Carthamustinctorius L. (Asteraceae); comparison of acidified chloral hydratesolution versus Example 1 solution;

FIG. 10 is a schematic representation of the chemical structures ofanalogs of 2,2,2-trichloroethanol useful for preparation of clearingagent and mounting solutions of the application;

FIGS. 11A-11H are representations of micrographs displaying fresh wholemounted specimens cleared with Example 1 clearing agent and mountingsolution. A-B: Basil leaf; C-F: Oregano leaf; G-H: Arabidopsis thalianaroot;

FIG. 12 is a representation of a micrograph of a red alga Polysiphoniasp gametophyte showing a secondary branch forming off the main axis,cleared with Example 1 clearing agent and mounting solution;

FIG. 13 is a representation of a micrograph of a roundworm free livingnematode Panagrellus redivivus (Animalia) anterior end showing internalstructures, cleared with Example 1 clearing agent and mounting solution;

FIG. 14 is a representation of a micrograph of a small aquaticcrustacean Daphnia sp. (Animalia) anterior end showing internalstructures, cleared with Example 1 clearing agent and mounting solution;

FIG. 15 is a representation of a micrograph of a characteristicDrosophila melanogaster (Animalia) compound eye, cleared with Example 1clearing agent and mounting solution;

FIG. 16 is a representation of a micrograph of a dorsal view of the headof Drosophila melanogaster (Animalia) showing compound eye, antenna andmouth parts, cleared with Example 1 clearing agent and mountingsolution;

FIG. 17 is a representation of a micrograph of the fungus Penicilliumsp. conidiophores with a chain of conidia (asexual spores) at the end,cleared with Example 1 clearing agent and mounting solution;

FIG. 18 is a representation of a micrograph of a basil downy mildew(Peronospora belbahrii), stained with iodine solution and sulfuric acid,cleared with Example 1 clearing agent and mounting solution;

FIG. 19 is a representation of a micrograph of a roundworm Panagrellusredivivus (Animalia.) stained with fuchsine, cleared with Example 1clearing agent and mounting solution;

FIG. 20 is a representation of a micrograph of characteristic starchgrains of Ginger (Zingiber officinale) stained with iodine solution,cleared with Example 1 clearing agent and mounting solution;

FIG. 21 is a representation of a micrograph of characteristic lignifiedfibers of Prunus africana stained with phloroglucinol/HCl solution,cleared with Example 1 clearing agent and mounting solution;

FIG. 22 is a representation of a micrograph of a mouse brain slicelabelled with NeuroTrace® 515/535 Yellow Fluorescent Nissl Stain; and

FIGS. 23A-23C are representations of micrographs of (a) human placentaltissue labelled with TO-PRO-3; (b) placental tissue labelled withanti-Ki67 antibody and detected with AlexaFluor488 secondary antibody;and (c) human placental tissue labelled with both.

DETAILED DESCRIPTION

Throughout this application, various patents, patent applications, andpublications are referenced. The disclosures of these patents, patentapplications, and publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art as known to those skilled therein as ofthe date of the invention described and claimed herein. The instantdisclosure will govern in the instance that there is any inconsistencybetween the patents, patent applications, and publications and thisdisclosure.

For convenience, certain terms employed in the specification, examples,and appended claims are collected here. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. The initial definition provided for a group or termherein applies to that group or term throughout the presentspecification individually or as part of another group, unless otherwiseindicated.

For the purposes of the present disclosure, the term “stain” or“staining” includes, but is not limited to, chemical interactionsbetween colored or fluorescent dyes and selected parts of a tissue; anymethod of allowing an antibody, aptamer, affibody, protein, peptide,single-stranded DNA or RNA, polysaccharide, glucosamine, dextran,lectin, or ligand, chemically conjugated to a fluorescent or coloredmolecule, to interact and bind to a tissue; any method of allowing anantibody, aptamer, affibody, protein, peptide, single-stranded DNA orRNA, polysaccharide, glucosamine, dextran, lectin, or ligand, chemicallyconjugated to a binding-site ligand (such as, but not limited to,biotin) to interact and bind to a tissue, and subsequent treatment witha protein conjugated to a colored or fluorescent molecule which bindsthe conjugated ligand with high affinity (such as, but not limited to,streptavidin); any method of allowing an antibody, aptamer, affibody,protein, peptide, single-stranded DNA or RNA, polysaccharide,glucosamine, dextran, lectin, or ligand, chemically conjugated to areactive enzyme to interact and bind to a tissue, which becomesoptically active upon subsequent treatment with the substrate of thereactive enzyme conjugated to a colored or fluorescent molecule; anymethod of allowing an antibody, aptamer, affibody, protein, peptide,single-stranded DNA or RNA, polysaccharide, glucosamine, dextran,lectin, or ligand, chemically conjugated to a reactive molecular moiety(such as, but not limited to, 1-ethynl) to interact and bind to tissue,and which becomes optically active upon subsequent treatment with acolored or fluorescent molecule conjugated to a molecular moiety (suchas, but not limited to, azido) that can react with the conjugate to forma covalent linkage; any method of allowing an antibody, aptamer,affibody, protein, peptide, single-stranded DNA or RNA, polysaccharide,glucosamine, dextran, lectin, or ligand, chemically conjugated to asingle strand or DNA/RNA or DNA/RNA derivatives, to interact and bindwith the tissue, and which becomes optically active upon subsequenttreatment with the antisense DNA/RNA/derivative conjugated to a coloredor fluorescent molecule.

It has now been discovered that aqueous solutions of trichloroethanol,and/or its derivatives or analogs, with certain other additives, caneffectively replace acidified chloral hydrate solution as a clearing andmounting medium for microscopy for both living and nonliving organismsand nonliving materials. These solutions can be used as non-chloralhydrate clearing and mounting compounds of appropriate refractive indexfor novel applications in microscopy. Given the current shortage andlimitation of access to the commercial universally-used clearing agent(acidified chloral hydrate), the present disclosure provides toconsumers and the general public a method that can replace the currentlyused clearing agent, and provides a method accessible to those who areno longer able to purchase the regulated compound chloral hydrate. Thus,the clearing reagents and methods of the present disclosure are ofimmediate commercial value and of significant impact because bothscientists and manufacturers have been seeking to find a replacement forchloral hydrate in microscopy and other optical applications. Theclearing reagents and methods of the present disclosure are also ofimmediate commercial value and of significant impact because they can beused with many stains as well as for semi-permanent and permanentmounting.

The present disclosure provides a substitute for acidified chloralhydrate glycerol solution, wherein the solution components, for exampletrichloroethanol, are inexpensive, easy to acquire, do not require a DEAlicense to possess and use, and greatly reduce the risk involved inoperations which formerly depended on the use of chloral hydrate. Thesolutions according to the disclosure possess a high refractive index(greater than or equal to about 1.3810; about 1.3810 to about 1.4880; orabout 1.4315 to about 1.4880. For example, it can be higher thanacidified chloral hydrate solution (1.4280), which results in clearer,and equivalent or higher quality viewing under a microscope. Highrefractive indices are required for clear viewing of objectives inmicroscopy, as materials with a high refractive index are moretransparent. As a reference, the refractive index of borosilicate(Pyrex) glass is 1.470. In more examples, the clearing agent andmounting solution of the disclosure have refractive indices higher thanthat of glass. It has been discovered that trichloroethanol, or itsderivatives or analogs, admirably meet the above-identified criteria asreplacements for chloral hydrate.

Use of the clearing agent and mounting solution of the disclosure helpsto macerate and digest clusters of cellular material, and helps toclarify and increase transparency of those tissues, minerals, elementsof interest in microscope slides. This solution is an effectiveimmersion medium, and useful in all types of fixative preparations andas an effective dehydration agent. The clearing compound and/or itsderivatives can also be used as a semi-permanent or permanent mount,allowing one to visualize specimens days or even months later. Thisclearing compound and/or its derivatives can also be used with manystains, allowing one to further visualize specimens and componentswithin specimens.

1. Clearing Agent and Mounting Solutions

This disclosure encompasses the identification of chemical compounds andcombinations thereof that have not heretofore been used in microscopyapplications. In one representation example, the clearing agent andmounting solution of the disclosure comprises any concentration of2,2,2-trichloroethanol, or derivatives or analogs thereof, and/or2,2,2-trichloroacetic acid, or derivatives or analogs thereof in waterand/or glycerol and/or alcohol solution, mixed for the purpose ofclearing and/or mounting media for microscope/optical use.

The clearing agent and mounting solution may alternatively comprise, inaddition to TCE and/or its derivatives, benzyl alcohol, benzyl benzoate,and/or benzyl ether. In addition, the clearing agent and mountingsolution described above may contain ethylene glycol (EG), polyethyleneglycol (PEG), and/or propylene glycol (PG), and/or derivatives thereofsuch as, but not limited to, glycol ethers, alkyl diols, alkyl triols,alkyl polyols, and other poly-alkoxy-hydroxy ethers, including, but notlimited to, diethylene glycol, dipropylene glycol, triethylene glycol,1,3-propanediol, 1,4-butanediol, 1,5-pentanediol,2(2-ethoxyethoxy)ethanol, bis(2-methoxyethyl) ether,2(2-butoxyethoxy)ethanol, PEG 200, PEG 400, and/or PEG 600.

For the purposes of the present disclosure, the term “derivative”encompasses a chemical compound which still retains the parent structureas a substructure, and can be chemically derived from the parent (e.g.,trichloroethanol or trichloroacetic acid). For example, with2,2,2-trichloroethanol as the parent, a derivative would retain the2,2,2-trichloroethoxy substructure, such as is found in thecorresponding acetate (1-acetoxy-2,2,2-trichloroethane, alternativelynamed 2,2,2-trichloroethylacetate) or the methyl ether(1-methoxy-2,2,2-trichloroethane) derivatives.

Also for the purposes of the present disclosure, the term “analog”encompasses a chemical compound in which the core structure of theparent is changed or further substituted, as is commonly known in themedicinal chemistry arts. For example, the change can includereplacement of atoms or groups with other atoms or groups (isosteres).Non-limiting examples of analogs of 2,2,2-trichloroethanol are displayedin FIG. 10.

The clearing agent and mounting solution of the disclosure comprisestrichloroethanol and/or its analogs or derivatives. It may also compriseboth trichloroethanol and/or its derivatives or analogs, andtrichloroacetic acid and/or derivatives or analogs. For example, theclearing agent and mounting solution may comprise about 5% to about 97%(v/v) trichloroethanol or derivatives thereof; 0% to about 50% (v/v)trichloroacetic acid; 0% to about 50% (v/v) glycerol; and optionally,water; wherein the refractive index of said clearing agent and mountingsolution is greater than or equal to about 1.3810, or about 1.3810 toabout 1.4880, or about 1.4315 to about 1.4880. The clearing agent andmounting solution can optionally further contain a C1-C6 alcohol,another acid, such as hydrochloric acid, and/or a dye or stain forvisualizing various components of the specimen. In one aspect,trichloroethanol is present in about 34.5% to about 97%. In anothernon-limiting example, trichloroacetic acid is present in 0% to about 5%.For example, glycerol can be present in about 0% to about 12.5%. Theglycerol can be supplemented or replaced entirely with one or morecompounds selected from the group consisting of ethylene glycol,propylene glycol, polyethylene glycols and/or derivatives thereof.

In other examples, the clearing agent and mounting solution comprisesabout 34.5% to about 97% trichloroethanol, 0% to about 5%trichloroacetic acid, and 0% to about 12.5% glycerol, or about 56.25%(v/v), trichloroacetic acid in about 1.25% (v/v), glycerol in about12.5% (v/v), methanol in about 25%, and water in about 5% (v/v).

A further example of the clearing agent and mounting solution of thedisclosure comprises trichloroacetic acid and/or derivatives withouttrichloroethanol and/or derivatives. One example of the clearing agentand mounting solution consists of an aqueous glycerol solution oftrichloroethanol and trichloroacetic acid (Example 1). Thetrichloroacetic acid can be supplemented or replaced entirely withhydrochloric acid and/or sulfuric acid. One example of the clearingagent and mounting solution consists of an aqueous glycerol solution oftrichloroethanol and hydrochloric acid. The 2,2,2-trichloroethanolanalog, 2-chloroacetaldehyde dimethyl acetal, FIG. 10, also serves as aneffective clearing agent. The clearing agent and mounting solution caninclude derivatives of 2,2,2-trichloroethanol and/or2,2,2-trichloroacetic acid created by adding/adjusting the concentrationof buffer, and/or acidic/basic additives intended to alter pH and/orsalt concentration of the solution; and/or compounds toincrease/decrease the digestive capacity of the reagent; and/or anyadditives intended to preserve specimens/samples; and/or any additivesintended for dying or staining applications.

With regard to the alcohol (other than trichloroethanol or derivatives),the clearing agent and mounting solutions can comprise one or more C1-C6alcohols, for example ethanol, 1-propanol, 2-propanol or t-butanol.

The clearing agent and mounting solutions can also further comprise adissolved plastic polymer, such as polyvinylpyrrolidone, polypropylene,polyethylene, polyether, polyamide; and/or monomeric units which aresubsequently polymerized in order to stabilize a mounting medium and/orspecimen; and/or one or more C1-C12 organic acids, such as formic acid,acetic acid, lactic acid, ascorbic acid, gallic acid, benzoic acid,toluic acid, p-hydroxybenzoic acid or citric acid; and/or a chemicalpreservative; and/or a salt of phosphate, citrate, acetate, tris, orother water-soluble buffers; the buffer can be added as an aqueoussolution; and/or alkaline hydroxide base, such as sodium hydroxide,calcium hydroxide or lithium hydroxide; the base can be added as anaqueous solution. The inventive clearing agent and mounting solutionand/or its derivatives and/or its analogs can also be used withcommercial and noncommercial stains just as effectively as currentclearing agents.

The present disclosure also encompasses various derivatives and/oranalogs of 2,2,2-trichloroethanol including mono- or poly-halogenatedbranched or unbranched alcohols, diols, glycols, aldehydes,aldehyde-hydrates, hemi-acetals, acetals, ketals, aminals, andhemi-aminals of at least 2 carbon units, where branches are defined asany mono- or poly-halogenated aliphatic or aromatic side chainscontaining hydroxyl, amino, ether, carboxyl, carboxyamido, carbonate,carbamyl, carbonyl-chloride, polyethyleneglycol, or aminoethanol groups,and any polymeric embodiment of such derivatives. Derivatives andanalogs of 2,2,2-trichloroacetic acid include mono- or poly-halogenatedbranched or unbranched carboxylic acids, carbamates, amides, andcarbonates of at least 2 carbon units, where branches are defined as anymono- or poly-halogenated aliphatic or aromatic side chains containinghydroxyl, amino, ether, carboxyl, carboxyamido, carbonate, carbamyl,carbonyl-chloride, polyethylene-glycol, or aminoethanol groups, and anypolymeric embodiment of such derivatives.

Other examples of the clearing agent and mounting solution comprisesabout 10% to about 97% (v/v) of a trichloroethanol derivative or analog;0% to about 50% (v/v) of a trichloroacetic acid derivative or analog; 0%to about 50% (v/v) of glycerol; and optionally, water; wherein therefractive index of said solution is greater than or equal to about1.3810; wherein the trichloroethanol derivative or analog is mono- andpoly-halogenated branched and unbranched alcohols, diols, glycols,aldehydes, aldehyde-hydrates, hemi-acetals, acetals, ketals, aminals,and hemi-aminals of at least 2 carbon units, and any polymericembodiment of such derivatives, and where branches are defined as anymono- or poly-halogenated aliphatic or aromatic side chains containinghydroxyl, amino, ether, carboxyl, carboxyamido, carbonate, carbamyl,carbonyl-chloride, polyethylene-glycol, or aminoethanol groups.

2. Optical Properties of the Solutions

The disclosure provides a solution which increases the apparenttransparency of an objective in microscopy/optical techniques byincreasing the refractive index of the medium in which said objective issuspended/immersed. Refractive index of a material is a dimensionlessquantity which represents the way light propagates through the material.The refractive index is defined as the factor by which the wavelengthand the velocity of the radiation with respect to in a vacuum. Therefractive index of a material is closely related to its dielectricconstant, and therefore to its transparency. The refractive index n of amaterial is given by the following equation:

${n = \sqrt{\frac{\sqrt{\epsilon_{1}^{2} + \epsilon_{2}^{2}} + \epsilon_{1}}{2}}},$where ε₁ and ε₂ represent the real and imaginary parts of the dielectricconstant, respectively. Materials which have a high dielectric constantcontain multiple lone pairs of electrons and/or electronegativeelements, which give them a high degree of polarizability, the propertywhich is expressed by the dielectric constant. A high degree ofpolarizability allows for an electromagnetic wave to propagate easilythrough the material, since as the electromagnetic wave propagatesthrough the material, it will induce a localized electromagnetic field.It is useful to use the analogy of waves through liquids, the lessviscous and easier the liquid is to move (by analogy related to higherpolarizability), the easier a wave can propagate through without losingenergy from absorption. In electromagnetic waves, a high polarizabilitycorresponds to this “easier movement” of the wave through the material,which results in less absorption of the wave by the material. Therefore,materials with high dielectric constants will have a low degree ofabsorption, and therefore a high degree of transparency, as theobjective light will make it through the material without absorptionloss. Effectively, the photons of light can escape more unscathed thanthey would in a material with a lower dielectric constant. And sincedielectric constant is related to refractive index as shown above,materials with a high refractive index will also be highly transparent,and therefore of great use in microscopy/optical techniques.

Refractive indices of exemplary clearing agent and mounting solutions ofthe disclosure are disclosed in Table 1.

TABLE 1 Refractive Clearing Agent and Mounting Solution Formulation¹Index Composition (v/v)% (nD20) TCE-34.5%; Glyc-12%; HCl (34%)-1.5%;H₂O-28%; 1.4155 MeOH-24% TCE-45%; Glyc-10%; HCl-1.25%; H₂O-23.75%;1.4310 MeOH-20% TCE-27.4%; Glyc-8.3%; TCAA-0.7%; MeOH-14.2%; 1.4370LA-49.4% TCE-56.25%; Glyc-12.5%; TCAA-1.25%; H₂O-30% 1.4315 TCE-27.4%;Glyc-8.3%; TCAA-0.7%; MeOH-14.2%; 1.4640 DMSO-49.4% TCE-94%; TCAA-5%;NaOH-1% 1.4880 TCE-91%; Glyc-3.3%; TCAA-0.3%; MeOH-5.4% 1.4875TCE-27.4%; Glyc-8.3%; TCAA-0.7%; MeOH-14.2%; 1.4770 Tol-49.4% TCAA-6%;NaOH-4%; H₂O-90% 1.4280 TCE-97%; TCAA-3% 1.4885 TCE-63.0%; Glyc-13.2%;TCAA-1.1%; MeOH-22.7% 1.4565 TCE-27.4%; Glyc-8.3%; TCAA-0.7%; MeOH-63.6%1.3895 TCE-27.4%; Glyc-8.3%; TCAA-0.7%; MeOH-14.2%; 1.4790 CWO-49.4%TCE-54.8%; Glyc-16.6%; TCAA-1.4%; MeOH-27.2% 1.4450 36% TCE + 36% BA +27% Glyc 1.5075 33% TCE + 33% BA + 33% Glyc 1.5021 33% TCE + 33% BA +17% Glyc + 17% BB 1.5202 29% TCE + 29% BA + 17% Glyc + 13% BB + 13% BE1.5236 42% TCE + 16.5% BA + 16.5% Glyc + 25% BE 1.5145 10% TCE + 90% BA1.5355 90% TCE + 10% BA 1.4951 10% TCE + 90% BB 1.5614 90% TCE + 10% BB1.4984 33% TCE + 33% BA + 33% BB 1.5331 10% TCE + 90% BE 1.5550 50%TCE + 50% BE 1.5255 90% TCE + 10% BE 1.4972 ¹TCE = trichloroethanol, CAA= trichloroacetic acid, Glyc = glycerol, MeOH = methanol, NaOH = sodiumhydroxide, LA = lactic acid, DMSO = dimethylsulfoxide, Tol = toluene,CWO = cedar wood oil. BA = benzyl alcohol BB = benzyl benzoate BE =benzyl ether

A number of analog structures have been provided which can be used as asubstitute for trichloroethanol (FIG. 10). 2-chloroacetaldehyde dimethylacetal has been shown to be effective at the same concentrations astrichloroethanol, although the refractive index is only 1.3810, at thelow end of the desired range. One way to achieve the desired refractiveindex is to incorporate a clearing agent having one or more halogens (F,Cl, Br, I) in a carbon skeleton which also contains a water solubilizinggroup capable of hydrogen bonding. For example, the carbon skeletons canbe selected from any mono- or poly-halogenated branched or unbranchedalcohol, diol, glycol, aldehyde, aldehyde-hydrate, hemi-acetal, acetal,ketal, aminal, or hemi-aminal of C1-C20 family, where branches aredefined as any mono- or poly-halogenated aliphatic or aromatic sidechains containing hydroxyl, amino, ether, carboxyl, carboxyamido,carbonate, carbamyl, carbonyl-chloride, polyethylene-glycol, oraminoethanol groups, and any polymeric arrangement of such derivatives.

In comparative qualitative examinations, the clearing agent and mountingsolutions of the disclosure perform as well as or better than acidifiedchloral hydrate. In quantitative examinations, the clearing agent andmounting solution of the disclosure has matched or outperformed thechloral hydrate-based solutions, and exhibited a refractive indexgreater than chloral hydrate solution, the universal standard. Thus, theclearing agent and mounting solution can be used to identify the sameanatomical characteristics or diagnostic features that are employed forthe identification of different plant, microbial, animal, and earthscience materials, without losing clarity, definition or resolution ofthe objective structures.

3. Methods

The clearing agent and mounting solutions of the disclosure are usefulfor microscopic identification of plants, plant parts, animals andmicrobial materials. The clearing agent and mounting solutions can beused for visualization of any living organisms such animals, fungi,protists, and bacteria, even with blood and plasma samples, as amounting medium in microscopy and/or other optical techniques withapplications in forensics, and biology and earth sciences. Thesesolutions can be used to clear specimens, rendering them transparent.These cleared specimens can later be differentially stained and highquality images obtained. Alternatively, uncleared specimens can bestained first and then cleared. The inventive clearing agent andmounting solutions and semi-permanent mounting media can also be usedwith non-living materials, including, but not limited to, soil particlesand geological samples.

The inventive clearing agent and mounting solutions are useful not onlyfor botanical microscopy but also for mycology, entomology, histology,food science, quality control (identification of living organisms formanufacture of pharmaceuticals, excipients, dietary products,adulterations, misidentifications, contaminations), forensics,nematology, virology, immunology, mineralogy, microscopy including, butnot limited to, differential interference contrast microscopy, electronmicroscopy, and other related applications of microscopy and optics.

For example, one application of the inventive clearing and mountingsolution is in quality assessment of commercial herbal products. It hasalso been determined that the inventive solutions are useful forclearing whole mounted dried, partially dry and fresh materials. Forexample, in basil, the oil glands, epidermis with stomata and underlyingpalisade cells could be observed (FIGS. 11A, B). In oregano, theepidermis over the vein with covering trichomes, capitates and peltateoil glands was distinguished (FIGS. 11C-F). Details of the cellularorganization of the root apical meristem in Arabidopsis thaliana can beobserved after clearing with this application (FIGS. 11G-H). Inaddition, a number of other herbs and spices (dry samples and wholetissues) were analyzed subsequently using the disclosure as clearingreagent with comparable results.

The solutions of the disclosure penetrate into tissues and render themmore transparent, as does acidified chloral hydrate solution. Aftertreatment with a clearing agent of the disclosure, samples are cleared,which allows internal as well as surface details to be easilyidentified. This feature is useful with whole mount tissues in whichdifferent layers of the transparent tissues are observed without theneed for sectioning or remounting. Clear tissues also allow for stainingtechniques to more effectively highlight diagnostic features in only onesingle step without requiring dehydration of the tissues orpre-treatment of the tissues (FIGS. 11A-H).

The inventive solutions are also effective to clear protists, animalsincluding, but not limited to, red algae (FIG. 12), round worms(Panagrellus redivivus, FIG. 13), water fleas, Daphnia sp (FIG. 14), andfruit flies (Drosophila melanogaster, FIGS. 15 and 16).

In order to identify different components in the cells, tissues can bestained. There are several staining combinations available to enhancethe details between different components of the cells. Stains areselected to provide the maximum contrast between particular structuresbased on their chemical composition. Much of the success of the imagesobtained after staining is due to the clearing agent used as apretreatment. The solutions of the present disclosure also have nointerference with the major stains used for particular structures. Asexamples, penicillium sp was first cleared with a clearing agent andthen stained with a solution of aniline blue in lactic acid (FIG. 17),downy mildew became dark brown after staining with iodine solution andsulfuric acid (FIG. 18), the round worm Pangrellus redivivus stainedpink/red with fuchsine (FIG. 19), starch grains were stained black withiodine solution in ginger rhizome dry samples (FIG. 20) and sclereidsfibers were stained red with phloroglucinol/HCl solution (FIG. 21).

The disclosure is also directed to a method of preparing a biologicalspecimen (e.g., plant, animal, etc.) for microscopy. One example of sucha method comprises applying a specimen to be examined to a microscopeslide, well, or cuvette; applying a sufficient quantity of the clearingagent and mounting solution to clear and mount said specimen; andoptionally, applying a cover slip. For example, about 1 drop to about100 drops, or about 2 drops of the clearing agent and mounting solutionare applied to the specimen on the slide in order to fix/mount thespecimen. The specimen can be further protected with a cover slip.

The disclosure also provides a method of preparing a specimen forspectrophotometric analysis. In this method a specimen is applied to acuvette or well, and a sufficient quantity of the clearing agent andmounting solution according to the disclosure is applied to clear thespecimen, e.g., about 1×-10× volume of sample.

In an alternative method, the specimen is stained before being cleared.More specifically, before soaking the specimen in a clearing solution, astain or dye is applied to the specimen to provide a stained specimen,whereby the features of the specimen are selectively highlighted. Thestained specimen is then soaked in the clearing solution according tothe disclosure for a sufficient amount of time to provide a cleared,stained specimen, e.g., at least about 1 minute to about 12 months. Thecleared, stained specimen is applied to a microscope slide, cuvette, orwell for observation, and optionally, is covered with a cover slip. Thestain or dye can be a fluorescent stain or dye, so that the cleared,stained specimen can be visualized using a fluorescent and/orepifluorescent and/or confocal microscope.

Reference will now be made to specific examples illustrating thedisclosure. It is to be understood that the examples are provided toillustrate exemplary embodiments and that no limitation to the scope ofthe disclosure is intended thereby.

EXAMPLES Example 1 Preparation of Plant Specimens

Plant materials were dried or used fresh. Dried plant materials wereground to a fine powder using a commercial coffee grinder.

A small quantity of fine powder material was spread on a microscopeslide, and mounted with two drops acidified chloral hydrate solution(control), or mounted with two drops of the clearing agent and mountingsolution of Example 1 and a cover slip was attached. Then the slide washeated on a hot plate (medium temperature) for 30 sec-60 sec until theclearing agent and mounting solution boiled. The microscopic analysiswas conducted using a Nikon eclipse 80i microscope, with the imagingsoftware NIS D 3.00 SP7. Differences or similarities in diagnosticfeatures or characteristics for each sample were recorded. Freshspecimens were submerged in the clearing agent and mounting solutionuntil they were transparent, usually taking around 20-30 minutesdepending the thickness of the material. Larger samples may require upto 2-3 days. Once the material was cleared, it was mounted on amicroscope slide with one or two drops of this disclosure and a coverslip was added. Fresh specimens were cleared and transparent, allowingthe visualization of deeper layers of tissues without losing clarity.

For staining the materials, first the material was cleared as describedabove, the cover slip was removed, one or two drops of the stain wasadded, and after a few minutes a cover slip was reapplied.

Example 2 Preparation of a Clearing Agent and Mounting Solution

4.5 mL of 2,2,2-trichlorethanol (Sigma-Aldrich, 99%, reagent grade) wasthoroughly mixed with 1.0 mL of glycerol. To this homogenous solutionwas added 2.5 mL of a 4% (m/v) aqueous solution of 2,2,2-trichloroaceticacid, at which time cloudiness was observed. The solution was mixedthoroughly until completely clear, about 5 min.

Example 3 Ginger (Rhizome), Zingiber officinale Roscoe (Zingiberaceae)

Powdered ginger samples are characterized by numerous fragments ofisodiametric thin-walled parenchyma cells containing starch granules;fragments of thin-walled fibers with oblique slit-like pits; fragmentsof scalariform, reticulate, and spiral vessels, thin-walled cells withsuberized radial walls; numerous starch granules with various forms suchas simple, flat, oval, oblong with terminal protuberance.

In ground ginger samples, fragments of parenchyma cells, cell withcircular striations (cork cells), fibers accompanied by vessels andabundant starch grains the compact epidermal cells with sharp edges canbe observed using the clearing agent and mounting solution of thedisclosure. There was no difference in the structures observed betweenthe inventive and standard clearing agent and mounting solutions.

FIGS. 1A-B show microscopic views of characteristic annular vesselelements and fibers of ginger: FIG. 1A, ginger characteristic annularvessel element and fibers using acidified chloral hydrate solution; FIG.1B, ginger characteristic annular vessel element with fibers andabundant starch grains attached to the fibers using clearing agent andmounting solution of Example 1.

FIGS. 2A-B show microscopic views of abundant starch grains of ginger.More starch grains can be observed using the clearing agent and mountingsolution of Example 1, versus acidified chloral hydrate solution: FIG.2A, ginger sample with characteristic abundant starch granules, mostlysimple, using acidified chloral hydrate solution; FIG. 2B, ginger samplewith abundant starch grains using clearing agent and mounting solutionof Example 1.

FIGS. 3A-B show microscopic views of ginger epidermis and parenchymacells. There is no difference in the structures observed using theinventive solution, versus acidified chloral hydrate solution: FIG. 3A,group of compact epidermal cells using acidified chloral hydratesolution; FIG. 3B, group of compact epidermal cells using clearing agentand mounting solution of Example 1.

Example 4 Mate (Leaves), Ilex paraguariensis (Aquifoliacea)

Ground Ilex leaves are characterized by upper epidermis composed bypolygonal cells with unevenly thickened walls. Lower epidermis cells aresmaller than those of the upper epidermis, and thinner cell walls,cuticular striations are well marked. Stomata anomocytic (epidermalcells surrounding the guard cell pair are not morphologically distinctfrom the other epidermal cells). Groups of lignified fibers are visible.

FIGS. 4A-B show microscopic views of fragments of epidermis over leafveins. The upper epidermis is composed of polygonal cells with unevenlythickened walls. Stomata are absent in the upper epidermis. There are nodifferences observed using the inventive solution, versus acidifiedchloral hydrate solution: FIG. 4A, fragment of polygonal cells of theupper epidermis over the vein using acidified chloral hydrate solution;FIG. 4B, fragments of polygonal cells of the upper epidermis over thevein using clearing agent and mounting solution of Example 1.

FIGS. 5A-B show microscopic views of the upper epidermis underlyingpalisade cells, large and closely packed. Circular striations can beobserved. Those cells treated with the invented solution are lessobscured versus those treated with acidified chloral hydrate solution:FIG. 5A, fragment of upper epidermis underlying parenchyma cells,cuticle is irregular striated, using acidified chloral hydrate solution;FIG. 5B, fragment of upper epidermis underlying parenchyma cells,cuticle is irregular striated, using clearing agent and mountingsolution of Example 1.

FIGS. 6A-B show microscopic views of fragments or groups of pericycle offibers. Fibers are lignified, moderately thickened and have pitted walls(gap in the internal secondary thickening of the cell wall). Pits fromthe fibers in FIGS. 6A and 6B can be clearly observed with both clearingagent and mounting solutions: FIG. 6A, longitudinal view of a veinsection showing fibers which have thickened walls with rounded or slitshaped pit (gap in the internal secondary thickening of the cell wall),using acidified chloral hydrate solution; FIG. 6B, longitudinal view ofa vein section showing fibers which have thickened walls with rounded orslit shaped pit (gap in the internal secondary thickening of the cellwall), using clearing agent and mounting solution of Example 1.

FIGS. 7A-B show microscopic views of the lower epidermis showingcharacteristic anomocytic stomata. There was no difference between theclearing agent and mounting solutions in clarity and function: FIG. 7A,lower epidermis surface showing anomocytic stomata and circularcuticular striations, using acidified chloral hydrate solution; FIG. 7B,lower epidermis surface showing anomocytic stomata and circularcuticular striations, using clearing agent and mounting solution ofExample 1.

Example 5 Safflower (Flower), Carthamus tinctorius L. (Asteraceae)

Powdered samples of flowers from Safflower have the abundant pollengrains with three noticeable germinal pores. The exine (outer coat ofthe pollen grain) is dentate and spinose. The presence of laticferousducts with a reddish brown secretion next to vessels was observed. Inground samples of safflower, pollen grains with three germinal pores,exine and laticiferous ducts with a reddish-brown secretion next tovessels could be observed.

FIGS. 8A-B show microscopic views of rounded or elliptical pollen grainswith three germinal pores, exine (outermost cell wall of pollen grain)dentate spinose: FIG. 8A, characteristic pollen grain with threegerminal pores, exine dentate, using acidified chloral hydrate solution;FIG. 8B, characteristic pollen grain with three germinal pores, exinedentate, using clearing agent and mounting solution of Example 1.

FIGS. 9A-B show microscopic views of laticiferous ducts (tubular cellscontaining latex fluid) with a reddish-brown secretion next to vesselselements. There were no differences observed using clearing agent andmounting solution, versus acidified chloral hydrate solution: FIG. 9A,two laticiferous ducts with a darker secretion next to the vesselelements, using acidified chloral hydrate solution; FIG. 9B,laticiferous duct filled with a darker secretion next to the vesselelements, using clearing agent and mounting solution of Example 1.

FIGS. 11A-H display fresh whole mounted plant specimens cleared withExample 1 clearing agent and mounting solution. FIGS. A-B: Basil leaf.FIG. A, epidermis with diacytic stomata, capitate and peltate glands;FIG. B: mesophyll cells with chloroplasts; FIGS. C-F: Oregano leaf; FIG.C, covering trichomes with thick cell walls over the vein and capitateglands; FIG. D, Close up of capitate glands (arrow); FIG. E: depictingepidermis and peltate oil gland; FIG. F: mesophyll cells; FIGS. G-H:Arabidopsis thaliana root; FIG. G, root tip cellular differentiation;FIG. H: xylem differentiation in root.

The clearing and mounting solution and its derivatives and/or analogscan also be used effectively in the same or a similar manner with cellsor tissues from animals including, without limitation, poultry, humans,livestock, reptiles, amphibians, insects and mites, as well as protists,mold, fungi, bacteria, and other microorganisms. For mammals,vertebrates, and invertebrates, specific portions can be prepared forvisualization, such as brain, spinal cord, skeletal system, etc.

The specimens displayed in FIGS. 12-21 were prepared analogously tothose above, using the clearing and mounting solution of Example 1.

FIG. 12 shows a red alga Polysiphonia sp gametophyte showing a secondarybranch forming off the main axis.

FIG. 13 shows round worm free living nematode Panagrellus redivivus(Animalia) anterior end showing internal structures (buccal cavity andjuveniles hatched internally).

FIG. 14 shows a small aquatic crustacean Daphnia sp. (Animalia) showinganterior section.

FIG. 15 shows characteristic Drosophila melanogaster (Animalia) compoundeye, showing numerous ommatidia (light detectors).

FIG. 16 shows a dorsal view of the head of Drosophila melanogaster(Animalia) showing compound eye, antenna and mouth parts.

FIG. 17 shows fungus Penicillium sp. conidiophores with a chain ofconidia (asexual spores) at the end.

FIG. 18 shows basil downy mildew (Peronospora belbahrii), protist, withdistinct staining of characteristic branched conidiophores and conidiaafter one week of inoculation. Stained with iodine solution and sulfuricacid.

FIG. 19 shows a round worm Panagrellus redivivus (Animalia) stained withfuchsine.

FIG. 20 shows characteristic starch grain of Ginger stained with iodinesolution.

FIG. 21 shows characteristic lignified fiber of Prunus africana stainedwith phloroglucinol/HCl solution.

Example 6 Mouse Specimens

A mouse was euthanized by cervical dislocation and immediately processedfor necropsy. The brain was removed and placed immediately into 10%neutral buffered formalin. It was then left at RT for approximately 3hr, and stored at 4° C. for 2 d. The brain was then sliced into about 3mm thick coronal sections, and one section was placed in 0.2% (v/v)Triton X-100 in phosphate buffered saline (PBS) at RT. After 5 d, thesection was transferred into about 3 mL PBS and washed twice for 10 mineach time. The section was then transferred to 1.0 mL PBS, and 30 μLNeuroTrace® 515/535 Yellow Fluorescent Nissl Stain (Molecular Probes,Eugene, Oreg.) and 1 μL TO-PRO®-3 (Molecular Probes, Eugene, Oreg.) wasadded. The specimen was then incubated in this solution at RT for about48 hr on an orbital shaker. Following incubation, the section wastransferred to 2.0 mL PBS for about 1 hr. The section was thentransferred into fresh 2.0 mL PBS and stored for 5 d. After storage, thesection was removed from PBS and the extra solution was removed with alow-lint wipe. The dried section was transferred into a solution of56.25% (v/v) TCE 1.25% (v/v) TCA, 12.5% (v/v) glycerol, 30% (v/v)methanol and then placed into a shaking incubator at 37 C. After about48 hr, the section was transferred to 100% glycerol and stored for 2 duntil imaged. Images were collected on a Leica SPIT confocal microscopeequipped with a HC PL APO 10×/0.40 objective and argon-krypton laser forexcitation at 488 nm, 534 nm, 594 nm, and 633 nm.

The image is shown in FIG. 22

Example 7 Human Specimens

Human placental cotyledon tissue, fixed with 10% neutral bufferedformalin and stored for over one year in fixative solution, wastransferred to PBS containing 0.01% (w/v) sodium azide for processinginto small pieces. A tissue piece was washed 3 times in PBS and twice in0.2% (v/v) Triton X-100 in PBS (PBST) for about 15 min each at RT. Thetissue was then transferred to PBST containing 20% (v/v) dimethylsulfoxide (DMSO) and 0.3 M glycine, and incubated for about 30 min at37° C. The tissue was then blocked with PBS containing 0.2% Tween-20,10% (v/v) DMSO and 6% (v/v) donkey serum for about 30 min at 37° C. Thetissue was then washed 3 times in PBS containing 0.2% Tween-20 and 10mg/ml heparin (PTwH) for about 15 min at 37° C. After washing, thetissue was then incubated for about 1 hr at 37° C. in a solution of 2.5μL Anti-Ki67 antibody (ab15580) (Abcam, Cambridge, Mass.) in 500 μL PBSwith 0.2% Tween-20, 5% (v/v) DMSO with 3% (v/v) donkey serum. Afterprimary antibody treatment, the tissue was then washed 10 times at RTwith PTwH for 5 min each. The tissue was then incubated for about 1 hrat 37° C. in a solution of 1 μL Goat Anti-Rabbit IgG H&L Alexa Fluor®488 (ab150081) (Abcam, Cambridge, Mass.) mixed with 1000 μL 3% (v/v)donkey serum in PTwH. After secondary antibody treatment, the tissue wasthen washed 10 times at RT with PTwH for about 5 min each. Afterwards,the tissue was washed 3 times in PBST for 15 min at 37° C. The tissuewas then incubated for about 30 min with a 1:1000 dilution of TO-PRO®-3(Molecular Probes, Eugene, Oreg.) in PBST at 37° C. The tissue was thenwashed 3 times with 50% (v/v) absolute ethanol in PBS for about 15 mineach at 37° C. The tissue was then washed 3 times with 70% (v/v)absolute ethanol in PBS for about 15 min each at 37° C. The tissue wasthen washed 3 times with absolute ethanol for about 15 min each at 37°C. Any remaining ethanol on the surface was dried with a low-lint wipe,and the tissue was transferred into a solution comprised of 33% (v/v)TCE, 33% (v/v) benzyl alcohol, and 33% (v/v) glycerol for 1 hr untilclear. The tissue was stored in this solution until ready to image.Images were collected on a Leica SPII confocal microscope equipped witha HC PL APO 10×/0.40 objective and argon-krypton laser for excitation at488 nm, 534 nm, 594 nm, and 633 nm.

The results are shown in FIGS. 23A-C.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific composition and procedures described herein. Such equivalentsare considered to be within the scope of this disclosure, and arecovered by the following claims.

The invention claimed is:
 1. A clearing agent and mounting solution,comprising: 5% to 97% (v/v) trichloroethanol or derivative thereof;16.5% to 36% (v/v) benzyl alcohol; and 12.5% to 50% glycerol,polyethylene glycol (PEG), propylene glycol (PG), ethylene glycol (EG),and/or derivatives thereof; and optionally water.
 2. The clearing agentand mounting solution of claim 1, comprising: 36% (v/v) oftrichloroethanol or derivative thereof; 36% (v/v) benzyl alcohol; and27% (v/v) glycerol.
 3. The clearing agent and mounting solution of claim1, comprising: 33% (v/v) of trichloroethanol or derivative thereof; 33%(v/v) benzyl alcohol; and 33% (v/v) glycerol.
 4. The clearing agent andmounting solution of claim 1, comprising: 33% (v/v) trichloroethanol orderivative thereof; 33% (v/v) benzyl alcohol; and 17% (v/v) glycerol;and further comprising: 17% (v/v) benzyl benzoate.
 5. The clearing agentand mounting solution of claim 1, comprising: 29% (v/v) trichloroethanolor derivative thereof; 29% (v/v) benzyl alcohol; and 17% (v/v) glycerol;and further comprising: 13% (v/v) benzyl benzoate; and 13% (v/v) benzylether.
 6. The clearing agent and mounting solution of claim 1,comprising: 42% (v/v) trichloroethanol or derivative thereof; 16.5%(v/v) benzyl alcohol; and 16.5% (v/v) glycerol; and further comprising:25% (v/v) benzyl ether.
 7. The clearing agent and mounting solution ofclaim 1, comprising: 36% (v/v) of trichloroethanol or derivativethereof; 36% (v/v) benzyl alcohol; and 27% (v/v) PEG, PG, EG, and/orderivatives thereof.
 8. The clearing agent and mounting solution ofclaim 1, comprising: 33% (v/v) of trichloroethanol or derivativethereof; 33% (v/v) benzyl alcohol; and 33% (v/v) PEG, PG, EG, and/orderivatives thereof.
 9. The clearing agent and mounting solution ofclaim 1, comprising: 33% (v/v) of trichloroethanol or derivativethereof; 33% (v/v) benzyl alcohol; and 17% (v/v) PEG, PG, EG, and/orderivatives thereof; and further comprising: 17% (v/v) benzyl benzoate.10. The clearing agent and mounting solution of claim 1, comprising: 29%(v/v) of trichloroethanol or derivative thereof; 29% (v/v) benzylalcohol; and 17% (v/v) PEG, PG, EG, and/or derivatives thereof; andfurther comprising: 13% (v/v) benzyl benzoate; and 13% (v/v) benzylether.
 11. The clearing agent and mounting solution of claim 1,comprising: 42% (v/v) trichloroethanol or derivative thereof; 16.5%(v/v) benzyl alcohol; and 16.5% (v/v) PEG, PG, EG, and/or derivativesthereof; and further comprising: 25% (v/v) benzyl ether.
 12. Theclearing agent and mounting solution of claim 1, wherein thetrichloroethanol derivative comprises mono- or poly-halogenated branchedor unbranched alcohols, diols, glycols, aldehydes, aldehyde-hydrates,hemi-acetals, acetals, ketals, aminals, and hemi-aminals of at least 2carbon units, wherein branches are defined as any mono- orpoly-halogenated aliphatic or aromatic side chains containing hydroxyl,amino, ether, carboxyl, carboxyamido, carbonate, carbamyl,carbonyl-chloride, polyethylene glycol, or aminoethanol groups, and apolymer thereof.
 13. The clearing agent and mounting solution of claim1, further comprising C1-C6 alcohol.
 14. The clearing agent and mountingsolution of claim 1, further comprising a solidification aid comprisingpolyethylene glycol, polyamide resin, polyvinylpyrrolidone, polyvinylalcohol, or mixtures thereof.
 15. A clearing agent and mountingsolution, comprising: 5% to 90% (v/v) trichloroethanol or derivativethereof; and 5% to 90% (v/v) benzyl alcohol.